ES2362021A1 - Method for obtaining a tissue-engineering product for regeneration of cartilaginous tissue - Google Patents

Abstract

Procedure for obtaining a tissue engineering product aimed at the regeneration of cartilage tissue. The present invention relates to a procedure for obtaining a tissue engineering product aimed at the regeneration of cartilage tissue, said product comprising expanded mesenchymal cells of bone marrow, a non-cellular matrix and a fibrin gel, the method comprising the steps of: (a) expanding the mesenchymal cells; (b) conjugating the mesenchymal cells to the matrix; (c) washing the product obtained in step (b); and (d) mixing the product obtained in step (c) with a fibrin gel.

Description

Procedure for obtaining a product of tissue engineering oriented to tissue regeneration cartilaginous

The present invention relates to a procedure for the preparation of an engineering product tissue-oriented regeneration of cartilaginous tissue articulate. More particularly, the present invention relates to a process for the preparation of a product comprising mainly mesenchymal cells, of bone origin, expanded, immobilized on supports and copolymerized and fixed in the area of implantation by means of fibrin gels and / or mechanical fit. He method of the present invention may further comprise a freezing stage of the tissue engineering product obtained, in order to have backup copies of it.

Due to the limited regeneration capacity of articular cartilage, defects caused by diseases degenerative or trauma represent a clinical problem not resolved. In this type of pathologies, the appearance of the lesions is associated with pain, loss of mobility and disability progressive associated with the appearance of arthritis or osteoarthritis. These circumstances have a serious impact on the quality of life of affected people.

The most common treatments of this type of lesions are mosaicoplasty, the autologous transplant of chondrocytes, heterologous osteochondral tissue transplantation, and abrasion or perforation arthroplasty. These techniques show as a common factor the contribution of specialized cells to the area injured to promote tissue regeneration / replacement damaged.

Among the treatments with the highest success rates is abrasion or perforation arthroplasty. This approach is based on inducing by bleeding the invasion of bone marrow mesenchymal cells from the subchondral bone towards the injured area, with the aim of repairing the lesion (Mitchell N et al. The resurfacing of adult rabbit articular cartilage by multiple perforations through the Bone Joint Surg Am . 1976; 58 (2): 230-3.24, Levy A et al. Chondral delamination of the knee in soccer players. Am J Sports Med . 1996; 24). The success of this technique depends on the correct integration, proliferation and differentiation of mesenchymal cells in the area to be repaired. Due to the great variability inherent in the technique, the result of this type of treatment is not very predictable. Consequently, repair is often obtained based on fibrocartilage instead of hyaline cartilage, which entails limitations in the quality and durability of therapy (Pelmari K et al. Do we really need cartilage tissue engineering ?. Swiss Med WKLY 2009; 139: 41-42; Kreuz PC et al. Results after microfracture of full-thickness chondral defects in different compartments in the knee. Osteoarthritis. Cartilage . 2006; 14 (11): 1119-25; Temenoff JS et al. Review: tissue engineering for regeneration of articular cartilage, Biomaterials, 2000; 431-40.

Therefore, there is a need to develop new technologies that solve the problems of the technique previous. The present invention discloses a new alternative Therapeutic based on tissue engineering. This approach is based on the use of a product that includes a matrix, based on synthetic or biomaterial materials, combined with cells and hydrogels that induces tissue regeneration damaged cartilaginous

The matrices used in the present invention must show particular characteristics that allow them to perform their support function for the reconstruction of the target tissue. These must be biocompatible, with adequate mechanical properties with respect to the implantation zone, with structural integrity and must be bioabsorbable. On the other hand, these matrices must be able to generate a biological environment that guarantees the contribution of nutrients to the cells to ensure that they can perform their regenerative function. To the previous characteristics it should be added that a matrix oriented to the regeneration of cartilaginous tissues should have the capacity to induce the differentiation of stem cells towards chondrocytes and that in this matrix this cell species is induced to manufacture tissue
cartilaginous

The other component of the engineering product tissue of the present invention, the cells, must also present a series of particular characteristics such as ease and security in obtaining it, come from a secure source from the cytogenetic point of view and have properties of multipotentiality, which allow their differentiation towards cells responsible for the production of cartilaginous tissue, the chondrocytes

Therefore, the present invention discloses a procedure for preparing a tissue engineering product Oriented to the regeneration of cartilage. This procedure is based on obtaining and using mesenchymal cells Expanded, autologous origin, from bone marrow. These cells expand and immobilize on supports based on synthetic or biomaterial polymers. and the set obtained is implanted and fixed in the area of injury by means of fibrin gels and / or mechanical fit

One of the advantages over Autograft-based treatments, such as chondroplasty or subchondral bleeding, is that using the prepared product by the process of the present invention it is not necessary subject the therapy recipient to one or more interventions of arthroscopic surgery for the extraction of biological material necessary for the regeneration of the damaged joint. He necessary cellular material is obtained by extracting bone marrow, which is performed on an outpatient basis. This approach, therefore, avoid potential complications arising from the obtaining autologous chondral material such as pain, bleeding, discomfort or complications due to infections.

Another advantage over the technique of abrasion or perforation arthroplasty applied to lesions of small size, which consists of injuring the subchondral bone to facilitate the contribution of regenerating cells, is that using the product prepared by the procedure herein invention a known cell dose can be applied and perfectly characterized, allowing treatment reproducible to patients undergoing this therapy.

Another additional advantage is that it is possible implant a product fully adapted to the topology of the injury.

In this aspect, the present invention discloses a method whereby the matrices are conjugated by a process of immobilization to a cellular product enriched in mesenchymal cells. Said cellular product with recognized chondroinductive capacity, is obtained by a selection and subsequent in vitro expansion of these cells. With the process of the present invention it is possible, by manipulating the process of cell expansion, to generate a wide range of quantities of cartilaginous graft that facilitates the treatment of lesions of different size and origin. This makes it applicable in a wide range of therapeutic applications, whose objective is the recovery of damaged cartilage.

Another feature of the procedure the present invention is that cell growth is carried carried out with supplements of culture media of human origin, which facilitates the rapid expansion of these cells, and avoids possible adverse effects arising from the contact of cells with non-human (or humanized) components. An advantage of The method of the present invention is that once the expansion, a cryopreservation of part of the cells with the aim of reserving doses to be used in Future treatments

On the other hand, the conjugation of the cells mesenchymal to biomatrices through a colonization process allows the location of cells with chondrogenic potential in The area to regenerate. This feature, different from others applications such as abrasion or perforation arthroplasty, ensures reparative cellular activity in the area to be treated and, to unlike other approaches to tissue engineering, it guarantees the permanence of the stem cells in the area of the lesion where They must do the repair work. Finally, the amalgamation of these particles with mesenchymal cells using fibrin gels allows to provide the set of plasticity and handling capacity facilitating in turn the immobilization of the mixture in the area to try to avoid the lack of structural coherence with the environment to regenerate.

The present invention discloses a procedure for obtaining a tissue engineering product oriented to the regeneration of cartilaginous tissue, comprising said product expanded bone marrow mesenchymal cells, a non-cellular matrix and a component based on hydrogels. Saying Procedure comprises the steps of:

(to)

expand cells mesenchymal;

(b)

conjugate mesenchymal cells to matrix;

(C)

wash the product obtained in the stage (b); Y

(d)

mix with a gel fibrin.

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The expansion of mesenchymal cells is performs in a manner known to those skilled in the art, with the objective of obtaining the amount of cells necessary to perform matrix conjugation, which will depend on the size of the lesion to try. Once the necessary amount has been obtained, the stage of conjugation of said cells to the matrix. This conjugation It can be done using agitated or non-agitated systems.

The mesenchymal cells obtained in the stage of expansion are resuspended in DMEM culture medium supplemented with serum at a concentration in the range of 1x103 to 1x10 7 cells per milliliter. If necessary you can they preserve said cells by cryopreservation to be used in the future.

Then the suspension is dispensed cell sterile in a bag or culture flask previously loaded with the matrix, which can be equipped with a pendular agitator, conjugate the mesenchymal cells to the matrix. The relationship between the number of cells per cubic centimeter is in the range of 1x102 to 1x108 cells per centimeter cubic matrix. Subsequently, it is allowed to incubate for 1 to 24 hours at a temperature of 35-38 ° C, saturation of CO2 of 2.5-7.5%, and higher relative humidity at 90% In the case of using agitation, the conditions of agitation used to ensure the anchoring of the cells are of 1 to 120 revolutions per minute.

Then the cell suspension mesenchymal immobilized on the matrix, obtained in the stage (b) wash several times using a physiological saline solution and it is sterilely dispensed in a conservation bag.

In step (d) of the process herein invention the product obtained in step (c) is mixed in a way sterile with a fibrin gel in a ratio of 0.1 to 10 units volumetric fibrinogen solution per each 0.1-10 volumetric units of thrombin solution and 0.1-10 volumetric units of colonized matrix, obtained in step (c).

The present invention will be described in Below with more details in reference to examples of realization. These examples, however, are not intended to limit the technical scope of the present invention.

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Examples Example 1 Preparation procedure of a cubic centimeter of tissue engineering product oriented to the regeneration of cartilage according to the present invention

12x10 6 mesenchymal cells were obtained of bone marrow by expansion using a culture medium free of animal serum and inoculated in a culture flask. Be used a medium based on DMEM supplemented with 10% human serum (v / v). The cell concentration was set at 6x105 cells per milliliter. The cell suspension was added sterile to a second bottle of plastic material equipped with a pendulum shaker, where 1 cubic centimeter of matrix was previously added. The 6x10 6 remaining cells were cryopreserved for the purpose of be able to perform new treatments, if necessary.

Incubation was performed in the following conditions: 37 ° C temperature, 5% CO2 saturation, and 95% relative humidity; then a cycle of 24-hour stirring at a speed between 120-200 rpm After this time, the product obtained was washed in order to eliminate cell debris and the culture medium. This operation was carried out using a physiological saline solution and the product obtained was packaged in a sterile bag

At the time of application of the product to the patient, an amalgamation of the matrices is performed with a fibrin gel. To perform this operation a volumetric ratio consisting of a solution unit of fibrinogen by a volumetric unit of thrombin solution and a volumetric unit of colonized bone matrix. Made the amalgamated the whole is given the form that allows it to adapt to the spatial distribution of the lesion and is located in the same.

While the invention has been described with respect to a preferred embodiment example, this should not be considered limiting of the present invention, which will be defined by the broader interpretation of the following claims.

Claims (7)

1. Procedure for obtaining a tissue engineering product oriented to tissue regeneration cartilaginous, said product comprising mesenchymal cells expanded bone marrow, a non-cellular matrix and a gel fibrin, the procedure comprising the steps of:

(to)

expand cells mesenchymal;

(b)

conjugate mesenchymal cells to matrix;

(C)

wash the product obtained in the stage (b); Y

(d)

mix the product obtained in the step (c) with a fibrin gel.

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2. Method according to claim 1, characterized in that step (b) is carried out by means of an agitated or non-agitated system. 3. Method according to claim 1 or 2, characterized in that it further comprises a stage of cryopreservation of the expanded mesenchymal cells for future use. Method according to any one of the preceding claims, characterized in that the ratio between the number of cells per cubic centimeter of matrix in step (b) is in the range of 1x102 to 1x108 cells per centimeter cubic matrix. 5. Method according to any of the preceding claims, characterized in that in stage (b) a CO2 saturation of 2.5-7.5 is carried out for 1 to 24 hours, at a temperature of 35-38 ° C. %, and a relative humidity greater than 90%. Method according to any one of the preceding claims, characterized in that in step (c) it is washed several times using a physiological saline solution. Method according to any one of the preceding claims, characterized in that the mixing with a fibrin gel of step (d) is carried out in a ratio of 0.1 to 10 volumetric units of fibrinogen solution per 0.1-10 volumetric units of thrombin solution and 0.1-10 volumetric units of colonized matrix, obtained in step (c).